2.1. Extracellular Matrix Receptors
Specific cell surface receptors (R) for extracellular matrix (ECM) components such as collagen, fibronectin and laminin have been described (reviewed by Hynes, 1987, Cell, 48:549–554; Hemler, 1988, Immunol. Today, 9:109). The functions of the extracellular matrix receptors (ECMRs I, II and VI) have been defined by affinity chromatography (Wayner and Carter, 1987, J. Cell Biol., 105:1873–1884; Staatz et al., 1989, J. Cell Biol., 198:1917–1924) and by preparing monoclonal antibodies that specifically inhibited the interaction of cells with purified ligands (Wayner and Carter, 1987, J. Cell Biol. 105:1873–1884) or ECM (Wayner et al., 1988, J. Cell Biol. 107:1881–1891).
A variety of ECMRs have been identified using these techniques. Using monoclonal antibodies, Wayner and Carter (1987, J. Cell Biol. 105:1873–1884) identified two classes of cell surface receptors for native collagen in human fibrosarcoma cells; class I was involved in cell adhesion to collagen, fibronectin and laminin, whereas class II was involved in cell adhesion only to native collagen. Wayner et al. (1988, J. Cell Biol. 107:1881–1891) identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6) and both collagen and fibronectin (P1B5); P1F8 and P1D6 were found to react with a 140 kD surface receptor known as ECMR VI. Kunicki et al. (1988, J. Biol. Chem. 263:4516–4519) reported that P1H5 (supra) also specifically inhibited adhesion of unactivated human platelets to collagen types I and III, but not to fibronectin. A complex comprising at least three glycoproteins was isolated from chicken embryo fibroblasts, using monoclonal antibodies which block cell adhesion to fibronectin (Knudsen et al., 1985, Exp. Cell Res. 157:218–226; Chen et al., 1985, J. Cell Biol. 100:1103–1114) whereas a complex of two glycoproteins was isolated from mammalian cells using vitronectin affinity chromatography (Pytela et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:5766–5770; Pytela et al., 1986, Science 231:1559–1562). Major platelet surface glycoproteins IIb and IIIa have been found to exist as a noncovalent 1:1 complex in the platelet membrane (Jennings and Phillips, 1982, J. Biol. Chem. 257:10458–10463) and to serve as an ECMR for fibrinogen (Bennett et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2417–2421; Marguerie et al., 1984, Eur. J. Biochem. 139:5–11), fibronectin (Gardner and Hynes, 1985, Cell 42:439–448; Plow et al., 1985, Blood 66:724–727), von Willebrand factor (Ruggeri et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:6038–6041) and vitronectin (Pytela et al., 1986, Science 231:1559–1562).
Structural homology is shared by the multitude of extracellular matrix receptors. The ECMRs are members of the integrin family of cell adhesion molecules and possess unique a subunits complexed to the integrin β1 subunit (Hynes, 1987, Cell 48:549–554; Wayner and Carter, 1987, J. Cell Biol. 105:1873–1884; Wayner et al., 1988, J. Cell Biol., 107:1881–1891). Additional members of the integrin receptor family include leukocyte adhesion proteins and the VLA antigens. The leukocyte adhesion proteins include LFA-1, Mac-1, and P150/95, and are dimeric glycoproteins composed of different α chains and a common, 95 kDa β chain, (Kishiomoto et al., 1987, Cell 48:681–690). VLA antigens are named for their very late appearance on cultured T lymphocytes (Hemler et al., 1983, J. Immunol. 131:334–340; Hemler et al., 1984, J. Immunol. 132:3011–3018; Hemler et al., 1985, Eur. J. Immunol. 15:502–50°). Antisera to the VLA-β subunit were found to block cell adhesion to fibronectin or laminin (Takada et al., 1987, Nature 326:607–610).
Interrelationships between these ECMRs have been identified. ECMR VI is identical to the prototype fibronectin receptor (Pytela et al., 1985, Cell, 40:191–198), α5β1, platelet glycoprotein (gp) Ic/IIa and VLA 5, ECMR II is identical to α2β1, platelet glycoprotein Ia/IIa and VLA 2 (Hemler et al., 1987, J. Biol. Chem., 262:11478–11485), and ECMR I is identical to α3β1 and VLA 3 (Kunicki et al., 1988, J. Biol. Chem., 263:4516–4519; Takada et al., 1988, J. Cellular Biochem., 37:385–393; Wayner et al., 1988, J. Cell Biol. 107:1881–1891). Monoclonal antibodies to α2β1, α3β1 and α5β1 (P1H5, P1D6 and P1B5) inhibit fibroblast or platelet adhesion to collagen, fibronectin and laminin-coated surfaces (Kunicki et al., 1988, J. Cell Biol. 107:1881–1891; Wayner et al., 1988, supra). Table I lists some of the members of the integrin family described supra, and Table II lists a number of monoclonal antibodies that recognize various ECMRs.
TABLE ITHE INTEGRIN RECEPTOR FAMILYSubunitKnownReceptorCompositionLigandsKnown FunctionsChicken Integrinα0β1FN, LM, VNCell adhesion,Complexα3β1Cell migration,CytoskeletalconnectionFibronectinα5β1FNAdhesion toreceptorFibronectinVitronectinα5β1VNAdhesion toreceptorVitronectinGlycoproteinαIIβ3FN, FB,Platelet adhesion andIIb/IIIaVN, VWFaggregationLFA-1α1β2ICAM-1,Leukocyte adhesion toICAM-2EndotheliumMAC-1αmβ2C3biC3b receptormonocyte andleukocyte adhesionp150/95α1–6β1C3biNeutrophil adhesionVLAs 1–6α1–6β1FN, COL,Cell adhesion,LAMmigration and Cyto-skeletal connectionEpithelialα6β4LAMEpithelial adhesionEpithelialαvβ5VN, FNEpithelial Celladhesion to VN, FNECMRs I,α2β1COL, LAMAdhesion to COL, LMII, VI, Vα3β1COL, LM, FNAdhesion to COL,α4β1FNLM, FNα5β1Previously unknownAdhesion to FN
TABLE IIANTI-ECMR ANTIBODIESAntibodyReceptorLigandReferenceP1H5α2β1Collagen(Wayner et al., 1987, J.LamininCell Biol. 105:1873–1884;Wayner et al., 1988, J. CellBiol. 107:1881–1891)P1B5α3β1Collagen(Wayner et al., 1987 J. CellFibronectinBiol. 105:1873–1884)P4C2α4β1Fibronectin(CS-1)P1D6α5β1Fibronectin(Wayner et al., 1988, J.Cell(Arg-Gly-Asp-Ser)Biol. 105:1873–1884)P4C10β1FN, COL, LAMP4119β2(CD18)
The β1 integrins are differentially expressed in cultured cells and tissue, and demonstrate clear differences in activation dependent expression. For example, expression of α5β1 in hematopoietic cells is restricted to subpopulations of thymocytes and peripheral blood lymphocytes, monocytes, acute lymphocytic or myelogenous leukemias, activated T cells, migrating hemopoietic precursor cells, and some cultured T, B or erythroleukemia cell lines (Bernardi et al., 1987, J. Cell Biol., 105:489–498; Cardarelli et al., 1988, J. Cell Biol., 106:2183–2190; Garcia-Pardo et al., 1989 Garcia-Pardo et al., 1989, Exp. Cell Res., 181:420–431; Giancotti et al., 1986, J. Cell. Biol., 103:429–437; Liao et al., 1987, Exp. Cell Res., 171:306–320; Wayner et al., 1988, J. Cell Biol. 107:1881–1891.
2.2. Fibronectin
Fibronectin is a protein found in the extracellular matrix as well as in plasma and on the surface of certain types of cells (Akiyama and Yamada, 1987, Adv. Enzymol. 59:1–57). In plasma, fibronectin exists as a glycoprotein heterodimer consisting of two similar subunits (called A and B chains), each having a molecular weight of approximately 220 kDa (Aidyama and Yamada, 1987, Adv. Enzymol. 59:1–57; Erickson et al., 1981, J. Cell Biol. 91:673–678). Multiple specialized intramolecular domains (Ruoslahti et al., 1981, J. Biol. Chem. 256:7277–7281) of the fibronectin molecule may be cleaved into fragments which, in turn, are capable of interacting with collagen, fibrin, heparin, and cell surfaces in a manner analogous to that of the intact molecule (Hynes and Yamada, 1982, J. Cell Biol. 95:369–377).
Cellular and plasma fibronectin heterodimers comprise similar but not identical polypeptides. The variability in the structure of fibronectin subunits derives from variations in fibronectin mRNA primary sequence due to alternative splicing in at least 2 regions of the pre-fibronectin mRNA (the ED and IIICS regions).
Fibronectin is capable of promoting adhesion of a variety of cell types, such as fibroblasts (Grinell et al., 1977, Exp. Cell Res. 110:175–210), macrophages (Bevilacque et al., 1981, J. Exp. Med. 153:42–60), polymorphonuclear leukocytes (Marino et al., 1985, J. Lab. Clin. Med. 105:725–730), platelets (Koteliansky et al., 1981, Fed. Euro. Biochem. Soc. 123:59–62) and keratinocytes (Clark et al., 1985, J. Invest. Dermatol. 84:378–383), to name but a few (Liao et al., 1989, Exp. Cell Res. 181:348–361). Interaction between fibronectin and a cell surface protein having a molecular weight of approximately 140 kDa has been observed in fibroblasts (Brown and Juliano, 1985, Science 228:1448–1451; Aidyama et al., 1986, J. Cell Biol. 102:442–448; Brown and Juliano, 1986, J. Cell Biol. 103:1595–1603; Wylie et al., 1979, J. Cell Biol. 80:385–402), endothelial cells (Plow et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:6002–6006), lymphoid cells (Brown and Juliano, 1986, J. Cell Biol. 103:1595–1603; platelets (Pytela et al., 1986, Science 228:1559–1562; Gardner and Hynes, 1985, Cell 42:439–448), muscle cells (Horowitz et al., 1985, J. Cell Biol. 101:2134–2144; Dambsky et al., 1985, J. Cell Biol. 100: 1528–1539; Chapman, 1984, J. Cell Biochem. 259:109–121), and osteosarcoma cells (Pytela et al., 1985, Cell 40:191–198).
The binding of fibronectin to cell surfaces may be competitively inhibited by fragments of fibronectin (Akiyama et al., 1985, J. Biol. Chem. 260:13256–13260). Using synthetic peptides, a sequence of what was thought to be the only minimal cell-recognition site was identified as the tetrapeptide Arg-Gly-Asp-Ser (RGDS) (SEQ ID NO: 1) Pierschbacher and Ruoslahti, 1984, Nature 309:30–33; Pierschbacher et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 80:1224–1227; Pierschbacher et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:5985–5988; Akiyama et al., 1985, J. Cell Biol. 102:442–448). The RGDS sequence present in the “cell binding” domain of fibronectin is the ligand for the prototype of fibronectin receptor described by Pytela et al. (1985, Cell 40:191–198).
Various observations suggested that regions other than RGDS may function in fibronectin binding (Humphries et al., 1986, J. Cell Biol. 103:2637–2647). For example, the binding affinity of synthetic peptides was found to be substantially lower than the binding affinity associated with larger fragments or intact fibronectin (Akiyama et al., 1985, J. Biol. Chem. 260:10402–10405; Akiyama et al., 1985, J. Biol. Chem. 260:13256–13260). McCarthy et al. (1986, J. Cell Biol. 102:179–188) reported binding affinity between a 33 kDa fragment of plasma fibronectin and B16-F10 melanoma tumor cells. Bernardi et al. (1987, J. Cell Biol. 105:489–498) reported that lymphoid precursor cells adhered to two different sites on fibronectin; the BaF3 cell line interacted with the RGD binding domain, whereas the PD31 cell line appeared to interact with a different domain located in the carboxy terminal segment and associated with a high affinity binding site for heparin.
Humphries et al. (1986, J. Cell Biol. 103:2637–2647) compared the ability of fibronectin fragments to form adhesive interactions with melanoma versus fibroblastic cells. Fibroblastic BHK cells were observed to spread rapidly on a 75 kDa fragment representing the RGDS containing cell-binding domain, whereas B16-F10 melanoma cells did not appear to spread on the 75 kDa fragment, but, instead were observed to spread on a 113 kDa fragment derived from the portion of fibronectin containing the type III connecting segment (CS) difference region, or V-region (in which alternative splicing of mRNA may occur). In this IIICS region, located near the fibronectin carboxyl terminus, the sequence Arg-Glu-Asp-Val (REDV) (SEQ ID NO: 2) appeared to have functional significance. Humphries et al. (1987, J. Biol. Chem. 262:6886–6892) of overlapping synthetic peptides spanning the IIICS region. Two nonadjacent peptides, CS1 and CS5, were found to be competitively inhibitory for adhesion of fibronectin to melanoma, but not to fibroblastic, cells, with CS1 showing greater inhibitory activity than CS5. Liao et al. (1989, Exp. Cell Res. 181:348–361), reported that MOPC 315, IgA-secreting lymphoid cells, in addition to binding to the cell binding domain via an RGD interaction, bound preferentially to the carboxy-terminal heparin binding domain by an RGD-independent mechanism. However, the adhesion sequence(s) present in the carboxy terminal regions of fibronectin and the cell surface receptor(s) responsible for adhesion of cells to these adhesion sequences have not been identified.
2.3. Biological Functions of Cell Adhesion Molecules
Adhesive interactions between cells have been found to occur during many important biological events, including tissue differentiation, growth and development, and also appear to play a critical role in the pathogenesis of various diseases (Humphries et al., 1986, J. Cell Biol. 103:2637–2647; Grinnell, 1984, J. Cell Biochem. 26:107–116; Hynes, 1986, Sci. Am. 254:42–51).
For example, adhesive interactions are known to be extremely important in the immune system; in which the localization of immune mediator cells is likely to be due, at least in part, to adhesive interactions between cells. Recirculation of lymphoid cells is non-random (Male et al., in “Advanced Immunology”, J. B. Lippincatt Co., Philadelphia, p. 14.4–14.5); lymphocytes demonstrate a preference for the type of secondary lymphoid organ that they will enter. In trafficing through a secondary lymphoid organ, lymphocytes must first bind to the vascular endothelium in the appropriate post-capillary venules, then open up the tight junctions between endothelial cells, and finally migrate into the underlying tissue. Migration of recirculating lymphocytes from blood into specific lymphoid tissues, called homing, has been associated with complementary adhesion molecules on the surface of the lymphocytes and on the endothelial cells of the high endothelial venules.
Likewise, the adherence of polymorphonuclear leukocytes to vascular endothelium is believed to be a key event in the development of an acute inflammatory response, and appears to be required for an effective chemotactic response as well as certain types of neutrophil-mediated vascular injury (Zimmerman and McIntyre, 1988, J. Clin. Invest. 81:531–537; Harlan et al., 1987, in “Leukocyte Emigration and its Sequelae”, Movat, ed. S. Karger A G, Basel, pp. 94–104; Zimmerman et al., ibid., pp. 105–118). When stimulated by specific agonist substances, the polymorphonuclear leukocytes Tonnensen et al., 1984, J. Clin. Invest. 74:1581–1592), endothelial cells (Zimmerman et al., 1985, J. Clin. Invest. 76:2235–2246; Bevilacque et al., J. Clin. Invest. 76:2003–2011), or both (Gamble et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:8667–8671) become adhesive; as a result, polymorphonuclear leukocytes accumulate on the endothelial cell surface.
In addition, studies with specific anti-glycoprotein antibodies in patients with immune deficits indicated that one or more components of the CD18 complex are required for effective neutrophil chemotaxis and other adhesion-related functions (Zimmerman and MacIntyre, 1988, J. Clin. Invest. 81:531–537). The CD18 complex is identical to the β2 integrin subfamily (supra).
During maturation and differentiation, lymphocyte sub-populations localize in different anatomical sites; for example, immature T cells localize in the thymus. Similarly, IgA-producing B cells are observed to localize in the intestinal mucosa (Parrott, 1976, Clin. Gastroenterol. 5:211–228). In contrast, IgG-producing B cells localize primarily in lymph nodes, from which IgG is secreted into the systemic circulation (Parrott and deSousa, 1966, Nature 212:1316–1317). T cells appear to be more abundant in skin epidermis than in mucosal linings (Cahill et al., 1977, J. Exp. Med. 145:420–428).
The physiologic importance of leukocyte adhesion proteins (supra) is underscored by the existence of a human genetic disease, leukocyte adhesion deficiency (LAD; Anderson et al., 1985, J. Infect. Dis. 152:668; Arnaout et al., 1985, Fed. Proc. 44:2664). Various studies have indicated that the molecular defect associated with LAD results in either lack of synthesis of the common β chain or normal rate of synthesis followed by rapid degradation (Liowska-Grospierre et al., 1986, Eur. J. Immunol. 16:205; Diamanche et al., 1987, Eur. J. Immunol. 17:417). In the severe form of LAD, neither LFA-1, Mac-1, nor p150/95 are expressed on the leukocyte membrane; low levels of leukocyte membrane expression have been observed in patients suffering from the moderate form of the disease. This leads to a defective mobilization of polymorphonuclear leukocytes and monocytes from the vasculature to the issues during the inflammatory response, with consequent recurrant bacterial infections (Anderson et al., J. Infect. Dis. 152:668; Arnaout et al., 1985, Fed. Proc. 44:2664).
ECMRs have also been observed to be associated with functions outside of the immune system. Loss of the IIb/IIIa platelet surface glycoprotein complex appears to result in defective platelet function in a genetic disease known as Glanzmann's thrombasthenia, (Hynes, 1987, Cell 48:549–554). Humphries et al. (1988, J. Cell Biol. 106:1289–1297) observed that neurons of the peripheral nervous system were able to extend neurites onto substrates bearing both the central cell-binding domain and the IIICS region of fibronectin. Furthermore, we have recently shown that neurite formation on laminin or fibronectin can be inhibited by antibodies to ECMRs.